Personalized medicine providing therapies, adapted to each patient's genetic predisposition (Roses 2002; Roses 2002A; Roses 2002B; Willard 2012; Lutz et al., 2011), is supported by the analysis of single nucleotide polymorphisms (SNPs) (Roses 2000). SNPs are single base variations and besides copy number variations the most abundant type of genetic variation found between members of one species (Shi 2001, Syvänen 2001; Venter et al., 2001). SNPs located in coding sequences can lead to structural and functional changes in the affected protein, enzymes or receptors. For example, the prothrombin G20210A mutation (PTM) is one of the most common genetic polymorphisms known to predispose to a first episode of venous thromboembolism (Marchiori et al., 2007). Most SNPs, however, are found in non-coding intergenic regions and often show no phenotypic effect. Intergenic SNPs present interesting markers for the determination of parentage (Hacia et al., 1999; Jorde et al., 2000), anthropology (Novembre et al., 2008; Schlebusch et al., 2012) or forensic tasks e.g. genetic fingerprinting. Many of these variations can affect predispositions for diseases or responses to drugs, chemicals and vaccines (Roses 2000; Kathiresan et al., 2009), which makes them especially interesting for pharmacogenomics (McCarthy and Hilfiker 2000; Relling and Dervieux 2001). The human genome project contributed to personalized medicine by identifying more than 2.4 million SNPs in 2001 (Lander et al., 2001; Venter et al., 2001). This created a basis for the first human haplotype map (HapMap) project with more than one million SNPs for which accurate and complete genotypes have been obtained in 269 DNA samples from four populations (International HapMap Consortium 2005). In a second step additional 2.1 million SNPs were added (Frazer et al., 2007). Phase III could further improve the quality with an extended set of 1,184 samples from 11 populations (International HapMap Consortium 2010). With the 1000 Genomes Project, a validated haplotype map of 38 million SNPs was published in 2012 (Consortium T1GP 2012). Genomes of 1,092 individuals sampled from 14 populations drawn from Europe, East Asia, sub-Saharan Africa and the Americas were analysed through a combination of low-coverage whole-genome sequence data, targeted deep exome sequence data and dense SNP genotype data (Consortium T1GP 2012).
It is most likely that SNP genotyping will be one of the future key technologies to diagnose these genetic variations between whole populations as well as in single patients. Different techniques can be used for the analysis of SNPs such as selective primer extension e.g. minisequencing (Pastinen et al., 2000; Syvänen 1999; Wartiovaara and Syvänen 2002), pyrosequencing (Ronaghi 2001) or allele specific amplification (ASA) (Myakishev et al., 2001; Pastinen et al., 2000; Shi, Bleavins and Ia iglesia 1999). Allele specific amplification (ASA) and selective primer extension (Pastinen et al., 2000) depend on the inefficient extension of a mismatched primer/template complex. Therefore highly selective DNA polymerases are urgently needed. Allele specific amplification through real-time PCR (ASA) allows detecting SNPs in a very efficient way. As unlike most other methods for SNP detection it does not require preliminary amplification of the target genetic material (Newton et al., 1989; Wu et al., 1989).
Another area of relevance is methylation-specific PCR (MSP). MSP is a widespread method used for the analysis of methylation patterns of cytosines at the C-5 position (5mC)—the most abundant DNA modification in vertebrates. 5-Methylcytosine is an important epigenetic mark and has a crucial role for activating or silencing genes (Ehrlich and Wang 1981; Jones 2012; Dawson and Kouzarides 2012). Methylation patterns alter during development and carcinogenesis (Feltus et al., 2006). Alterations of epigenetic marks like DNA methylation play a crucial role in the onset of diseases like cancer and therefore are important early stage cancer biomarkers (Dawson and Kouzarides 2012; Feltus et al., 2006, Heichmann and Warren 2012; Heyn and Esteller 2012; Deng, Liu and Du 2012). In general, it appears that the detection of epigenetic alterations is a promising and emerging tool for cancer diagnostics, prognostics, and prediction of response to therapies that awaits broad application in the future. In combination with genetic mutation analysis, epigenetic analysis will result in a very powerful approach along these lines for early diagnostics of cancer and other diseases such as neuro-developmental and metabolic disorders as well as auto-immune diseases (Heyn and Esteller 2012; Deng, Liu and Du 2012; Deng, Liu and Du 2010).
MSP, as well ASA, is a very cost-effective method that does not require specialized equipment and can be performed in almost any laboratory. Selectivity is the most important factor in ASA and MSP and can be increased by the use of modified primer probes (Strerath et al., 2002; Strerath and Marx 2005; Johnson 2004) or by the employment of mutated DNA polymerases possessing a higher mismatch extension selectivity as compared to the wild type enzyme. A selective polymerase would enable reliable ASA and MSP without the need of any substrate modifications and can thus be the most cost and work efficient solution.
Several DNA polymerases with increased DNA replication fidelity are known (Suzuki et al., 2000), e.g. for the Klenow fragment of E. coli DNA polymerase I and the thermostable Thermus aquaticus (Taq) DNA polymerase (Summerer et al., 2005) or the Pyrococcus furiosus (Pfu) DNA polymerase (Biles and Connolly 2004). It is known that the exchange of amino acids, affecting the interaction between polymerase and primer/template complex or the binding pocket's geometry, can lead to a change in selectivity of DNA polymerases (Kunkel and Bebenek 2000). In previous studies for instance, an increase in Taq DNA polymerase selectivity by changing the polar amino acids Q and H (Gln, and His) of motif C into two unipolar amino acids (Q to L and H to L) was achieved. Motif C is highly conserved in the palm domain within the family A, B, X and Y polymerases and plays a role in the identification of mismatched bases in the primer/template complex (Loh and Loeb 2005; Franklin, Wang and Streitz 2001). While discovered with Taq DNA polymerase, a member of family A, in further studies (Rudinger, Kranaster and Marx 2007) this concept to increase mismatch extension selectivity could be transferred to the B family Pfu DNA polymerase. The respective amino acids are found in the highly conserved motives YGDTD and KXY in eukaryotes, bacteria, archaea and viruses (Blasco et al., 1995).
Along this line, WO 2005/074350 describes DNA polymerases of the family A, with increased mismatch discrimination and therefore increased selectivity.
US2012/0258501 describes a Thermus sp. Z05 DNA polymerase, which can tolerate a 3′ primer mismatch.
WO 2011/157435 describes DNA polymerase with increased 3′ mismatch discrimination.
DE 10 2006 025 153 describes DNA polymerase with increased mismatch discrimination.
Therefore, the technical problem underlying the present invention can be seen in the provision of alternative DNA polymerases with increased mismatch discrimination. In other words, the DNA polymerases of the present invention have increased selectivity.
The technical problem is solved by the embodiments reflected in the claims, described in the description, and illustrated in the Examples and Figures.
Surprisingly, it has been found that by mutation of basic amino acids (arginine and lysine) that are in direct contact to the phosphate backbone of the primer strand in the closed conformation of the Klenow fragment of the Taq polymerase (KlenTaq) leads to the generation of DNA polymerases with advantageous properties which make the DNA polymerases of the present invention particularly suitable for, e.g. various diagnostic applications.
Specifically, the present inventors demonstrated that the selectivity of a DNA polymerase can be altered by substituting a polar amino acid residue that interacts with the backbone of the primer strand. They identified interesting mutants with increased mismatch selectivity for each examined amino acid position.
More specifically, the present inventors found that the DNA polymerase mutants described herein are suitable for diagnostic purposes, while, however, uses of the DNA polymerases described herein are not limited to diagnostic purposes. By way of example, the inventors selected the mutant (R660V) which showed best performance on genomic DNA templates to demonstrate its high mismatch selectivity. In order to further speed up detection, the inventors set-up a multiplexing assay with both allele-specific primers present in one reaction followed by an allele-identification melting curve readout. With this the SNP detection is possible in one single reaction well, as at least the presence of one allele-specific amplificate is serving as a positive control indicating both a sufficient template quality and polymerase activity. Hence, multiplexing applications are a preferred embodiment of the methods and uses of the present invention. Additionally, in particular KlenTaq R660V is able to perform ASA in the presence of whole blood sample with no previous DNA purification. This fast and easy system allows SNP genotyping in multiwell format in less than two hours with minimal costs, circumventing time and cost intensive sample preparations. However, the present invention also provides other DNA polymerase mutants that have similar or even identical properties like KlenTaq R660V. Finally they could show that the DNA polymerases described herein, in particular, KlenTaq R660V, is suitable for MSP.
The above being said, the present invention relates to a DNA polymerase, comprising an amino acid sequence comprising at one or more positions corresponding to position(s) 487, 508, 536, 587 and/or 660 of the amino acid sequence of the Taq polymerase shown in SEQ ID NO:1 or at corresponding positions of a Klenow fragment thereof, an amino acid substitution.
The present invention also relates to a DNA polymerase, comprising an amino acid sequence comprising at one or more positions corresponding to position(s) 210, 231, 259, 310 and/or 383 of the amino acid sequence of the KlenTaq polymerase shown in SEQ ID NO:2 an amino acid substitution.
Additionally, the present invention relates to the use of the DNA polymerase of the present invention, for detection of at least one SNP, which SNP is comprised in a target sequence.
The present invention further relates to the use of the DNA polymerase of the present invention, for detection of a methylation status of a target sequence.
The present invention also relates to the use of a DNA polymerase of the present invention, for discrimination between as matched and a mismatched primer, wherein said primers hybridize to a target sequence, and wherein the mismatched primer comprises a non-canonical nucleotide in a position of up to seven bases from its 3′ end in relation to the target sequence to which it hybridizes.
The present invention further relates to the use of a DNA polymerase of the present invention as a diagnostic for diagnosing a disease of a subject, which disease is associated with a SNP or a methylation status of a target sequence, which target sequence is comprised in the genomic DNA of the subject.
In addition, the present invention relates to the use of a DNA polymerase of the present invention, in the presence of a dye that binds to double stranded DNA at a concentration of 10×-60× e.g., SYBRGreenI
The present invention further relates to the use of a DNA polymerase of the present invention in the presence of blood.
The present invention also relates to the use of a DNA polymerase of the present invention, at a concentration of 25 nM-600 nM.
Additionally, the present invention relates to a DNA polymerase for use in in vitro diagnosis of a disease of a subject, which disease is associated with a SNP in a target sequence, which target sequence, is comprised in the genomic DNA of a subject.
Also, the present invention relates to a DNA polymerase for use in in vitro diagnosis of a disease of a subject, which disease is associated with a methylation status of a target sequence, which target sequence is comprised in the genomic DNA of a subject.
The present invention further relates to an in vitro method for detecting at least one SNP in at least one template comprising contacting the DNA polymerase of the present invention with                i) the at least one template;        ii) at least one matched and/or at least one mismatched primer,                    wherein said primers hybridize to the target sequence, and            wherein the mismatched primer comprises a non-canonical nucleotide in a position of up to seven bases from its 3′ end in relation to the target sequence to which it hybridizes; and                        iii) nucleoside triphosphates.        
In addition, the present invention relates to an in vitro method for detecting at least one methylated nucleotide, preferably cytosine in at least one template comprising contacting the DNA polymerase of the present invention with                i) the at least one template;        ii) at least one matched and/or at least one mismatched primer,                    wherein said primers hybridize to the target sequence, and            wherein the mismatched primer comprises a non-canonical nucleotide in a position of up to seven bases from its 3′ end in relation to the target sequence to which it hybridizes; and                        iii) nucleoside triphosphates.        
Further, the present invention relates to a nucleic acid molecule coding for a DNA polymerase of the present invention.
Also, the present invention relates to a vector comprising the nucleic acid of the present invention.
The present invention additionally relates to a host cell comprising the vector of the present invention and/or the nucleic acid of the present invention.
The present invention also relates to a kit comprising the DNA polymerase of the present invention.
Further, the present invention relates to a kit-of-parts comprising the DNA polymerase of the present invention.